The Combination of Particle Irradiation With the Hedgehog Inhibitor GANT61 Differently Modulates the Radiosensitivity and Migration of Cancer Cells Compared to X-Ray Irradiation

Abstract

Due to the advantages of charged particles compared to conventional radiotherapy, a vast increase is noted in the use of particle therapy in the clinic. These advantages include an improved dose deposition and increased biological effectiveness. Metastasis is still an important cause of mortality in cancer patients and evidence has shown that conventional radiotherapy can increase the formation of metastasizing cells. An important pathway involved in the process of metastasis is the Hedgehog (Hh) signaling pathway. Recent studies have demonstrated that activation of the Hh pathway, in response to X-rays, can lead to radioresistance and increased migratory, and invasive capabilities of cancer cells. Here, we investigated the effect of X-rays, protons, and carbon ions on cell survival, migration, and Hh pathway gene expression in prostate cancer (PC3) and medulloblastoma (DAOY) cell lines. In addition, the potential modulation of cell survival and migration by the Hh pathway inhibitor GANT61 was investigated. We found that in both cell lines, carbon ions were more effective in decreasing cell survival and migration as well as inducing more significant alterations in the Hh pathway genes compared to X-rays or protons. In addition, we show here for the first time that the Hh inhibitor GANT61 is able to sensitize DAOY medulloblastoma cells to particle radiation (proton and carbon ion) but not to conventional X-rays. This important finding demonstrates that the results of combination treatment strategies with X-ray radiotherapy cannot be automatically extrapolated to particle therapy and should be investigated separately. In conclusion, combining GANT61 with particle radiation could offer a benefit for specific cancer types with regard to cancer cell survival.

Keywords: GANT61; Hedgehog pathway; carbon ion; gene expression; migration; particle therapy; proton; radiosensitization.

Figure 1

Colony survival assay of PC3 and DAOY cells irradiated with X-rays, protons or carbon ions. Surviving fraction of PC3 (A) and DAOY (B) cells after irradiation with different doses, calculated 14 days after start of colony survival. Means ± SEM of three independent experiments performed in triplicate.

Figure 2

Migration of PC3 and DAOY cells after X-ray, proton, or carbon ion irradiation. The Boyden chamber assay was performed in order to assess the migratory potential of PC3 (A) and DAOY (B) cells. The migration assays was started 24 h after different doses of X-rays, protons, or carbon ions. Means ± SEM of three independent experiments performed in triplicate. ^*p ≤ 0.05 vs. control cells; ^**p ≤ 0.01; ^***p ≤ 0.001; ^****p ≤ 0.0001.

Figure 3

Effect of GANT61 in combination with radiation on the survival of PC3 cells. Colony survival curves of PC3 cells pre-treated for 72 h with GANT61 (10μM) and irradiated with different doses of X-rays (A), protons (B), or carbon ions (C). Means ± SEM of three independent experiments performed in triplicate.

Figure 4

Effect of GANT61 in combination with radiation on the survival of DAOY cells. Colony survival curves of DAOY cells pre-treated for 72 h with GANT61 (10μM) and irradiated with different doses of X-rays (A), protons (B), or carbon ions (C). Means ± SEM of three independent experiments performed in triplicate. ^*p ≤ 0.05 vs. control cells; ^**p ≤ 0.01.

Figure 5

Migration of PC3 cells after the combination of GANT61 with irradiation. The Boyden chamber assay was performed in order to assess the migratory potential of PC3 cells after the combination of GANT61 (72 h pre-incubation; 10μM) with X-rays or with carbon ions. Means ± SEM of 2–3 independent experiments performed in triplicate. ^*p ≤ 0.05 vs. control cells; ^**p ≤ 0.01; ^***p ≤ 0.001; ^****p ≤ 0.0001.

Figure 6

Migration of DAOY cells after the combination of GANT61 with irradiation. The Boyden chamber assay was performed in order to assess the migratory potential of DAOY cells after the combination of GANT61 (72 h pre-incubation; 10μM) with X-rays or with carbon ions. Means ± SEM of 2–3 independent experiments performed in triplicate. ^*p ≤ 0.05 vs. control cells; ^**p ≤ 0.01; ^***p ≤ 0.001; ^****p ≤ 0.0001.